Amit Kumar Tripathi

Transbilayer translocation of membrane phosphatidylserine and its role in macrophage invasion in Leishmania promastigotes.

Infectivity of Leishmania promastigotes has been shown to be growth cycle-dependent and restricted to the stationary phase. By using annexin V-FITC binding and procoagulant activity measurement assays, we show here that the promastigotes in the stationary phase contain significantly higher amounts of phosphatidylserine (PS) on their surface as compared to the log phase promastigotes. We also demonstrate that the infectivity of the promastigotes is determined by the presence of PS on their surface. In addition, by using NBD-labelled phospholipids, we show that the promastigote plasma membrane contains ATP-dependent out-to-in and ATP-independent in-to-out PS translocases which regulate the PS localisation in two-halves of the membrane bilayer, and that the greater amounts of external PS observed in the stationary phase promastigotes is perhaps due to the slower ATP-dependent out-to-in PS movements in these cells, as compared to the log phase promastigotes.

MicroRNA: a new and promising potential biomarker for diagnosis and prognosis of ovarian cancer.

Epithelial ovarian cancer (EOC) is the leading cause of death among all gynecological malignancies. Despite the technological and medical advances over the past four decades, such as the development of several biological markers (mRNA and proteins biomarkers), the mortality rate of ovarian cancer remains a challenge because of its late diagnosis, which is specifically attributed to low specificities and sensitivities. Under this compulsive scenario, recent advances in expression biology have shifted in identifying and developing specific and sensitive biomarkers, such as microRNAs (miRNAs) for cancer diagnosis and prognosis. MiRNAs are a novel class of small non-coding RNAs that deregulate gene expression at the posttranscriptional level, either by translational repression or by mRNA degradation. These mechanisms may be involved in a complex cascade of cellular events associated with the pathophysiology of many types of cancer. MiRNAs are easily detectable in tissue and blood samples of cancer patients. Therefore, miRNAs hold good promise as potential biomarkers in ovarian cancer. In this review, we attempted to provide a comprehensive profile of key miRNAs involved in ovarian carcinoma to establish miRNAs as more reliable non-invasive clinical biomarkers for early detection of ovarian cancer compared with protein and DNA biomarkers.

Attenuated neuroprotective effect of riboflavin under UV-B irradiation via miR-203/c-Jun signaling pathway in vivo and in vitro.

BackgroundRiboflavin (RF) or vitamin B2 is known to have neuroprotective effects. In the present study, we report the attenuation of the neuroprotective effects of RF under UV-B irradiation. Preconditioning of UV-B irradiated riboflavin (UV-B-RF) showed attenuated neuroprotective effects compared to that of RF in SH-SY5Y neuroblostoma cell line and primary cortical neurons in vitro and a rat model of cerebral ischemia in vivo.ResultsResults indicated that RF pretreatment significantly inhibited cell death and reduced LDH secretion compared to that of the UV-B-RF pretreatment in primary cortical neuron cultures subjected to oxygen glucose deprivation in vitro and cortical brain tissue subjected to ischemic injury in vivo. Further mechanistic studies using cortical neuron cultures revealed that RF treatment induced increased miR-203 expression which in turn inhibited c-Jun expression and increased neuronal cell survival. Functional assays clearly demonstrated that the UV-B-RF preconditioning failed to sustain the increased expression of miR-203 and the decreased levels of c-Jun, mediating the neuroprotective effects of RF. UV-B irradiation attenuated the neuroprotective effects of RF through modulation of the miR-203/c-Jun signaling pathway.ConclusionThus, the ability of UV-B to serve as a modulator of this neuroprotective signaling pathway warrants further studies into its role as a regulator of other cytoprotective/neuroprotective signaling pathways.

Single Amino Acid Substitutions at Specific Positions of the Heptad Repeat Sequence of Piscidin-1 Yielded Novel Analogs That Show Low Cytotoxicity and In Vitro and In Vivo Antiendotoxin Activity

ABSTRACT Piscidin-1 possesses significant antimicrobial and cytotoxic activities. To recognize the primary amino acid sequence(s) in piscidin-1 that could be important for its biological activity, a long heptad repeat sequence located in the region from amino acids 2 to 19 was identified. To comprehend the possible role of this motif, six analogs of piscidin-1 were designed by selectively replacing a single isoleucine residue at a d (5th) position or at an a (9th or 16th) position with either an alanine or a valine residue. Two more analogs, namely, I5F,F6A-piscidin-1 and V12I-piscidin-1, were designed for investigating the effect of interchanging an alanine residue at a d position with an adjacent phenylalanine residue and replacing a valine residue with an isoleucine residue at another d position of the heptad repeat of piscidin-1, respectively. Single alanine-substituted analogs exhibited significantly reduced cytotoxicity against mammalian cells compared with that of piscidin-1 but appreciably retained the antibacterial and antiendotoxin activities of piscidin-1. All the single valine-substituted piscidin-1 analogs and I5F,F6A-piscidin-1 showed cytotoxicity greater than that of the corresponding alanine-substituted analogs, antibacterial activity marginally greater than or similar to that of the corresponding alanine-substituted analogs, and also antiendotoxin activity superior to that of the corresponding alanine-substituted analogs. Interestingly, among these peptides, V12I-piscidin-1 showed the highest cytotoxicity and antibacterial and antiendotoxin activities. Lipopolysaccharide (12 mg/kg of body weight)-treated mice, further treated with I16A-piscidin-1, the piscidin-1 analog with the highest therapeutic index, at a single dose of 1 or 2 mg/kg of body weight, showed 80 and 100% survival, respectively. Structural and functional characterization of these peptides revealed the basis of their biological activity and demonstrated that nontoxic piscidin-1 analogs with significant antimicrobial and antiendotoxin activities can be designed by incorporating single alanine substitutions in the piscidin-1 heptad repeat.

Simultaneous determination of centchroman and tamoxifen along with their metabolites in rat plasma using LC–MS/MS

AIM Tamoxifen and centchroman are two non-steroidal, selective estrogen receptors modulators, intended for long term therapy in the woman. Because of their wide spread use, there is a possibility of co-prescription of these agents. MATERIALS & METHODS We studied the probable pharmacokinetic interaction between these agents in breast cancer model rats. A simple, sensitive and rapid LC-ESI-MS/MS method was developed and validated for the simultaneous determination of tamoxifen, centchroman and their active metabolites. RESULTS The method was linear over a range of 0.2-200 ng/ml. All validation parameters met the acceptance criteria according to regulatory guidelines. CONCLUSION LC-MS/MS method for determination of tamoxifen, centchroman and their metabolites was developed and validated. Results show the potential of drug-drug interaction upon co-administration these two marketed drugs.

A study of ER stress in rat model of cerebral venous sinus thrombosis

Cerebral venous sinus thrombosis (CVST) is a rare form of stroke. The role of endoplasmic reticulum (ER) stress markers is well documented in arterial stroke but has not been evaluated in venous stroke. The present study has been undertaken to investigate the role of ER stress in rodent model of CVST. For inducing CVST, a cranial window was made to expose superior sagittal sinus (SSS). A strip of filter paper soaked with 40% ferric chloride was applied on exposed cranial window while in sham operated control 0.9% saline was used. Clinical evaluations were done on day 1, 2, and 7 for neurological deficit. Rota rod test and brain infarction volume were also measured. Brain tissue was collected from infarcted portion for further analysis using real time polymerase chain reaction and western blot technique for ER stress markers. Augmented expression of ER stress markers and up regulation of apoptotic genes were found in infarcted tissue. These markers improved on day 7. It is concluded that ER markers are up regulated at an early stage of CVST and may be important in pathophysiology.

Identification of GXXXXG motif in Chrysophsin-1 and its implication in the design of analogs with cell-selective antimicrobial and anti-endotoxin activities

Marine fish antimicrobial peptide, chrysophsin-1 possesses versatile biological activities but its non-selective nature restricts its therapeutic possibilities. Often small alterations in structural motifs result in significant changes in the properties of concerned proteins/peptides. We have identified GXXXXG motif in chrysophsin-1. Glycine residue(s) of this motif in Chrysophsin-1 was/were replaced with alanine, valine and proline residue(s). Of these, proline-substituted Chrysophsin-1 analogs exhibited significantly reduced cytotoxicity towards mammalian cells. Further, these analogs showed broad-spectrum activity against Gram-positive, Gram-negative bacteria, Methicillin-resistant Staphylococcus aureus strains and fungi and also retained antibacterial activity in presence of physiological salts, serum and at elevated temperatures indicative of their therapeutic potential. These Chrysophsin-1 analogs also inhibited lipopolysaccharide (LPS) induced pro-inflammatory responses in THP-1 cells and in murine primary macrophages. One of these single proline-substituted Chrysophsin-1 analogs inhibited LPS-stimulated pro-inflammatory cytokine production in BALB/c mice and elicited appreciable survival of mice administered with a lethal dose of LPS in a model of severe sepsis. The data for the first time showed the implication of GXXXXG motifs in functional and biological properties of an antimicrobial peptide and could be useful to design novel anti-microbial and anti-endotoxin peptides by employing this motif.